Summary: Cardiac injury is increased in the aged population following ischemia-reperfusion (IR). Age- related mitochondrial dysfunction contributes to cardiac injury in aged hearts during IR through inhibiting bio-energy production, increasing production of reactive oxygen species and increasing susceptibility to opening of the mitochondrial permeability of transition pore (MPTP). The purpose of the present project is to identify the molecular mechanisms that contribute to mitochondrial dysfunction in aged heart mitochondria. Calpain 1 (CPN1) is a calcium-dependent cysteine protease that exists in both cytosol and mitochondria. Our ongoing study identified that mitochondrial localized calpain 1 (mit-CPN1) exists in intermembrane space and matrix compartments. Activation of the mit-CPN1 led to the damage of complex I and the degradation of metabolic enzymes including pyruvate dehydrogenase (PDH). We hypothesize that oxidative stress generated by the age-impaired electron transport chain will activate mit-CPN1 leading to proteolytic damage in the matrix in the baseline state in aged hearts. Some complex I subunits are also oriented toward the matrix. We will use a proteomic approach coupled with bioinformatic analysis to identify key targets of mit-CPN1 including cleavage of complex I subunits, PDH, and unknown matrix peptides. These metabolic defects in aged hearts will augment cardiac injury by facilitating complex I damage and MPTP opening during IR. Knockout of calpain 4 (a regulatory subunit of calpain 1 and 2) eliminates calpain 1 and calpain 2 activities. Cardiac specific calpain4 knockout mice that result in mitochondria devoid of mit-CPN1 are used as a molecular model to identify authentic mit-CPN1 targets. Calpain 1 knockout mice are also used to rule out the potential effect of calpain 2 activation. Adult and aged C57BL/6 mice are studied. Myocardial infarction and the sensitivity of the MPTP opening are determined in mitochondria isolated from mouse hearts following in vitro or in vivo IR. Mitochondrial functions are measured. Characterization of the role of the mit-CPN1 activation in cardiac injury during IR will guide to develop novel pharmacological strategies to attenuate cardiac injury and subsequent heart failure in the aged population following IR.